Objective To analyze the gene expression characteristics of chronic rhinosinusitis with nasal polyps (CRSwNP) using bioinformatics methods, aim to investigate the potential biomarkers and their diagnostic value of CRSwNP. Methods (1) The CRSwNP Gene expression data set was downloaded from the American Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) between CRSwNP patients and healthy controls were screened through data analysis. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the identified DEGs. Protein-protein interaction (PPI) networks were constructed utilizing the STRING database, and the key genes were identified by using the cytoHubba plugin. The "Cibersort" package was used to analyze the influence of key genes on common immune cells. (2) Thirty-two patients diagnosed with CRSwNP in the First Affiliated Hospital of Hebei North University from June 2022 to June 2023 were selected as the CRSwNP group, and 21 patients with simple deviation of nasal septum without a history of sinusitis during the same period were selected as control group. The pathological characteristics of specimens in the two groups were examined using hematoxylin-eosin (HE) staining. Immunohistochemistry and Western blotting were used to detect the expression levels of key genes in CRSwNP. The levels of key proteins in plasma were detected using ELISA, and ROC curve was used to analyze its efficacy in diagnosing CRSwNP. Results (1) Analysis of three gene expression database sets (GSE36830, GSE23552, and GSE194282) showed that there were 156 DEGs in CRSwNP. GO functional enrichment and KEGG pathway analysis indicated that the functions of the above DEGs were mostly related to immune functions. Key genes such as cytochrome b-245 β chain (CYBB) and colony-stimulating factor 1 receptor (CSF1R) were identified. (2) The results of HE staining revealed that the epithelial of CRSwNP tissue was metaplastic into stratified squamous epithelium with interstitial edema. Both immunohistochemistry and Western blotting analyses indicated that the expression levels of CYBB and CSF1R in the CRSwNP group were significantly increased compared to control group (P<0.05). ELISA results demonstrated that CYBB [(21.20±3.00) μg/ml vs. (17.66±1.66) μg/ml, P<0.05] and CSF1 [(477.37±86.63) pg/ml vs. (370.71±66.24) pg/ml, P<0.05] in CRSwNP group were significantly increased compare to control group. ROC curve analysis showed that plasma concentrations of CYBB and CSF1 had AUCs of 0.888 (95%CI 0.802-0.974) and 0.821 (95%CI 0.711-0.931) for diagnosing of CRSwNP, respectively; their combined AUC was 0.927 (95%CI 0.851-1.000). Conclusions CYBB and CSF1R may be involved in the occurrence and development of CRSwNP. Plasma CYBB and CSF1 have high diagnostic value for CRSwNP.